Acute or severe hemolysis result from the release of significant amounts of toxic free heme leading to organ, tissue and cellular damage manifested in a variety of pathological conditions such as acute hemolytic transfusion reactions (AHTR). In the field of transfusion medicine, AHTR, while rare, is a clinically significant concern. AHTR-mediated RBC hemolysis releases hemoglobin (Hb) into the vasculature which promotes a pro-oxidative and pro-inflammatory environment. Additionally, free heme is exquisitely nephrotoxic leading to renal failure through a number of mechanisms including oxidative damage and heme deposition in the renal tubules. Given the highly destructive properties of cell free heme, development of an inhibitor to detoxify its oxidative properties remains a major unmet medical need. Our laboratory has developed peptide constructs known as Peptide Inhibitors of Complement C1 (PIC1) that inhibit antibody-initiated, classical complement pathway mediated destruction of human RBC in vitro as well as in a rat model of AHTR. Recently, we have discovered that PIC1 can inhibit the peroxidase activity of RBC lysates and purified human Hb in in vitro assays via an antioxidant mechanism mediated by two cysteine residues contained within the PIC1 sequence. Given its dual ability to inhibit complement-mediated RBC destruction and neutralize Hb peroxidase activity, PIC1 provides an exceptional opportunity to mitigate the AHTR disease process. The experimental focus of this project is to assess inhibition of heme peroxidase toxicity of PIC1 in our well-defined rat model as well as define the mechanism by which PIC1 exerts its inhibitory effect on Hb with the long range goal of producing a therapeutic molecule for clinical testing in humans. Our specific hypothesis is that PIC1 will inhibit Hb peroxidase activity in vivo via its reactive cysteine residues, thus limiting toxic free radical formation and inflammation minimizing downstream sequelae, including kidney failure and death. Specific Aim 1 will evaluate the efficacy of PIC1 in preventing heme-mediated tissue and organ injury created by infusing RBC lysates in the AHTR rat model. Following optimization of the amount of RBC lysates required to induce a reliable phenotype, prophylactic and rescue administration of PIC1 in the animal model will be assessed. Readouts will consist of various blood chemistry markers as well as kidney damage evaluated by histopathology. Specific Aim 2 will evaluate the mechanism of PIC1 peroxidase inhibitory activity. PIC1 acts as an antioxidant to inhibit peroxidase activity of Hb via a redox mechanism involving the two cysteine residues. We will evaluate the role each of these cysteine residues play in this activity in vitro and in vivo. First we will utilize derivatives of PIC1 in which one or both cysteine residues are substituted to assess Hb peroxidase inhibitory/antioxidant activity in vitro. Secondly, we will determine the efficacy of selected PIC1 derivatives on inhibition of heme toxicity in the animal model. Successful accomplishment of these aims will establish PIC1 as a novel therapeutic to detoxify free heme in the setting of acute hemolysis such as AHTR, hemolytic anemia and sickle cell disease.